Menniti F S, Miller R N, Putney J W, Shears S B
Calcium Regulation Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1993 Feb 25;268(6):3850-6.
There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.
尽管肌醇1,3,4,5,6 - 五磷酸酯和六磷酸酯是最丰富的肌醇多磷酸酯，但关于它们在细胞内功能的信息却很少。目前认为它们作为抗氧化剂（格拉夫，E.，马奥尼，J. R.，布莱恩特，R. G.，和伊顿，J. W.（1987年）《生物化学杂志》259卷，3620 - 3624页）或“管家”分子（伯里奇，M. J.，和欧文，R. F.（1989年）《自然》341卷，197 - 205页）发挥被动作用的观点，源于对它们代谢惰性的看法。然而，我们发现，在与5 mM氟化物和5 mM ATP一起孵育的细胞匀浆中，肌醇六磷酸酯（Km = 2 ± 0.5 microM，Vmax = 9 ± 2 pmol/毫克蛋白质/分钟）和肌醇1,3,4,5,6 - 五磷酸酯（Km = 13 ± 4 microM，Vmax = 11 ± 5 pmol/毫克蛋白质/分钟）都被转化为了极性更强的产物。在用0.5 - 20 mM氟化物处理的完整细胞中也观察到了这些反应，并且通过比较氟化物对用[3H]肌醇进行不同标记的细胞在短期或脉冲追踪实验中的影响，证实了前体/产物关系。由于这些新产物能被烟草焦磷酸酶和碱性磷酸酶相对特异性地水解，所以确定它们是肌醇焦磷酸酯。这些焦磷酸酯被细胞匀浆迅速代谢回它们的五磷酸酯和六磷酸酯前体。这种内源性焦磷酸酶活性在体外被5 mM氟化物抑制高达99%。在用10 mM氟化物孵育的完整细胞中，在1小时内，约20%的肌醇1,
